Molecular Mechanisms of Cell Migration Project summary: Recognition of extracellular matrix (ECM) cues is critical to many developmental and homeostatic processes, and misinterpretation of cues underlies diseases such as metastasis. Cells probe for ECM cues by extending actin-based protrusions to bind ECM and form anchors to help them move forward. However, little is known about the decision processes and signaling pathways that convert ECM probing by protrusions into directed cell migration. Many conventional microscopy studies posit that protrusions are uniformly covered with randomly diffusing unbound ECM receptors that stochastically bind ECM. With our new approach of dense- field single molecule super-resolution microscopy, we have discovered a concentrated band of unbound ECM receptors at the front of protrusions that have characteristic molecular patterns of receptor organization, mobility, and functionalization. These patterns depend on both cellular protrusive activity and specific receptor cytoplasmic binding domains, indicating that cytoplasmic interactions, visible only at the single molecule level regulate how unbound integrins are organized in preparation for probing for ECM. In this proposal we will define the cellular decision processes that convert ECM probing into directed cell migration by integrating molecular measures of the organization, interaction, and functionalization of populations of ECM receptors with cellular measures of the assembly of adhesions, actin regulatory pathways, and protrusive activity. We will: 1) Define mechanisms that create the global spatial-temporal patterns of unbound ECM receptor organization, mobility, and conformation at the front of cell protrusions that regulate the functionalization of protrusions to probe ECM; 2) Identify signaling pathways that bound ECM receptors use to separate the decision to convert an ECM cue into directed migration from the decision to continue to probe the ECM through actin cytoskeletal dynamics. These studies will directly contribute to our understanding of cell recognition of ECM cues in many development and homeostasis processes, as well as our understanding of the misinterpretation of cues, which underlies diseases such as metastasis.